Bacillus thuringiensis isolate active against dipteran pests

ABSTRACT

Disclosed and claimed is a novel Bacillus thuringiensis isolate designated B.t. PS192N1 which has dipteran activity. Thus, this isolate, or mutants thereof, can be used to control such insect pests. Further, genes encoding novel  delta -endotoxins can be removed from the isolate and transferred to other host microbes, or plants. Expression of the  delta -endotoxins in such hosts results in the control of susceptible insect pests in the environment of such hosts.

This application is a continuation of Ser. No. 07/777,828, filed Oct.19, 1991, now abandoned, which is a divisional of Ser. No. 07/596,829,filed Oct. 12, 1990, now abandoned.

BACKGROUND OF THE INVENTION

Many hundreds of strains of Bacillus thuringiensis (B.t.) produceinsecticidal toxins designated as delta endotoxins. They are synthesizedby sporulating B.t. cells. When toxin is ingested by a susceptibleinsect, the cells of the gut epithelium are destroyed.

The reported activity spectrum of B.t. covers insect species within theorders Lepidoptera and Coleoptera, many of which are major pests inagriculture and forestry. The activity spectrum also includes the insectorder Diptera, which includes mosquitoes and black flies. See Couch, T.L. (1980) "Mosquito Pathogenicity of Bacillus thuringiensis var.israelensis," Developments in Industrial Microbiology 22:61-76; Beegle,C. C. (1978) "Use of Entomogenous Bacteria in Agroecosystems,"Developments in Industrial Microbiology 20:97-104. Dipteran insects areserious nuisances as well as being vectors of many serious human andanimal diseases such as malaria, onchocerciasis, equine encephalitis,and dog heartworm.

The two varieties of B.t. known to kill mosquitos and blackflies areB.t. israelensis (B.t.i.) (Goldberg, L. J., J. Margalit [1977] MosquitoNews 37:355-358) and B.t. morrisoni (B.t.m.) (Padua, L. E., M. Ohba, K.Aizawa [1984] J. Invertebrate Pathology 44:12-17). These are not harmfulto non-target organisms (Mulla, M. S., B. A. Federici, H. A. Darwazeh[1982] Environmental Entomology 11:788-795), and play an important rolein the integrated management of dipteran pests. They are safe to use inurban areas, and can be used in aquatic environments without harm toother species.

Dipteran pests are a major problem in the poultry and cattle industries.The horn fly, a serious cattle pest, is killed by B.t. in the larvalstages (Temeyer, K. B. [1990] "Potential of Bacillus thuringiensis forfly control," Fifth International Colloquium on Invertebrate Pathologyand Microbial Control, Society for Invertebrate Pathology, 352-356).

There are also dipteran pests of plants, such as Hessian fly, Medfly,and Mexfly, for which a B.t. product would be very valuable.

BRIEF SUMMARY OF THE INVENTION

The subject invention concerns a novel Bacillus thuringiensis isolatewhich has activity against dipteran pests.

Specifically, the invention comprises a novel B.t. isolate designatedB.t. PS192N1, and mutants thereof, and novel delta endotoxin genesobtainable from this B.t. isolate which encode proteins which are activeagainst dipteran pests.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 is a photograph of a 12% SDS polyacrylamide gel of B.t.i.,B.t.m., and B.t. PS192N1.

DETAILED DISCLOSURE OF THE INVENTION

The novel Bacillus thuringiensis isolate of the subject invention hasthe following characteristics:

Characteristics of B.t. PS192N1

Colony morphology--large colony, dull surface, typical B.t.

Vegetative cell morphology--typical B.t.

Culture methods--typical for B.t.

Flagellar serotype--19, tochigiensis

Inclusion--amorphic

Alkali-soluble proteins--SDS polyacrylamide gels show 140, 122, 76, 72,and 38 kilodalton proteins.

Insecticidal activity--B.t. PS192N1 has an LC₅₀ of 10 μg protein/mlsuspension against Aedes aegypti.

                  TABLE 1                                                         ______________________________________                                        Comparison of the proteins in the known dipteran-active                       strains B.t.i. and B.t.m. with B.t. PS192N1                                                    Molecular Weight of Proteins                                 Strain           (SDS-PAGE) kDa                                               ______________________________________                                        B.t. israelensis 130, 67, 27                                                  B.t. morrisoni   138, 128, 125, 67, 27                                        B.t. PS192N1 (tochigiensis)                                                                    140, 122, 76, 72, 38                                         ______________________________________                                    

The novel B.t. isolate of the invention, and mutants thereof, can becultured using standard known media and fermentation techniques. Uponcompletion of the fermentation cycle, the bacteria can be harvested byfirst separating the B.t. spores and crystals from the fermentationbroth by means well known in the art. The recovered B.t. spores andcrystals can be formulated into a wettable powder, a liquid concentrate,granules or other formulations by the addition of surfactants,dispersants, inert carriers and other components to facilitate handlingand application for particular target pests. The formulation andapplication procedures are all well known in the art and are used withcommercial strains. The novel B.t. isolate, and mutants thereof, can beused to control dipteran pests.

The culture of the subject invention was deposited in the AgriculturalResearch Service Patent Culture Collection (NRRL), Northern RegionalResearch Center, 1815 North University Street, Peoria, Ill. 61604 USA.

    ______________________________________                                        Culture          Accession No.                                                                             Deposit date                                     ______________________________________                                        Bacillus thuringiensis PS192N1                                                                 NRRL B-18721                                                                              October 5, 1990                                  ______________________________________                                    

The subject culture has been deposited under conditions that assure thataccess to the culture will be available during the pendency of thispatent application to one determined by the Commissioner of Patents andTrademarks to be entitled thereto under 37 CFR 1.14 and 35 U.S.C. 122.The deposit is available as required by foreign patent laws in countrieswherein counterparts of the subject application, or its progeny, arefiled. However, it should be understood that the availability of thedeposit does not constitute a license to practice the subject inventionin derogation of patent rights granted by governmental action.

Further, the subject culture deposit will be stored and made availableto the public in accord with the provisions of the Budapest Treaty forthe Deposit of Microorganisms, i.e., it will be stored with all the carenecessary to keep it viable and uncontaminated for a period of at leastfive years after the most recent request for the furnishing of a sampleof the deposit, and in any case, for a period of at least thirty (30)years after the date of deposit or for the enforceable life of anypatent which may issue disclosing the culture. The depositoracknowledges the duty to replace the deposit should the depository beunable to furnish a sample when requested, due to the condition of thedeposit. All restrictions on the availability to the public of thesubject culture deposit will be irrevocably removed upon the granting ofa patent disclosing it.

The toxin genes harbored by the novel isolate of the subject inventioncan be introduced into a wide variety of microbial hosts. Expression ofthe toxin gene results, directly or indirectly, in the intracellularproduction and maintenance of the pesticide. With suitable hosts, e.g.,Pseudomonas, the microbes can be applied to the situs of dipteraninsects where they will proliferate and be ingested by the insects. Theresult is a control of the unwanted insects. Alternatively, the microbehosting the toxin gene can be treated under conditions that prolong theactivity of the toxin produced in the cell. The treated cell then can beapplied to the environment of target pest(s). The resulting productretains the toxicity of the B.t. toxin.

Where the B.t. toxin gene is introduced via a suitable vector into amicrobial host, and said host is applied to the environment in a livingstate, it is essential that certain host microbes be used. Microorganismhosts are selected which are known to occupy the "phytosphere" oraquatic environment. These microorganisms are selected so as to becapable of successfully competing in the particular environment (cropand other insect habitats) with the wild-type microorganisms, providefor stable maintenance and expression of the gene expressing thepolypeptide pesticide, and, desirably, provide for improved protectionof the pesticide from environment degradation and inactivation.

A large number of microorganisms are known to inhabit the phylloplane(the surface of the plant leaves), and/or the rhizosphere (the soilsurrounding plant roots), and/or aquatic environments. Thesemicroorganisms include bacteria, algae, and fungi. Of particularinterest are microorganisms, such as bacteria, e.g., genera Bacillus,Pseudomonas, Erwinia, Serratia, Klebsiella, Xanthomonas, Streptomyces,Rhizobium, Rhodopseudomonas, Methylophilius, Agrobacterium, Acetobacter,Lactobacillus, Arthrobacter, Azotobacter, Leuconostoc, Alcaligenes andClostridium; fungi, particularly yeast, e.g., genera Saccharomyces,Cryptococcus, Kluyveromyces, Sporobolomyces, Rhodotorula, andAureobasidium; microalgae, e.g., families Cyanophyceae,Prochlorophyceae, Rhodophyceae, Dinophyceae, Chrysophyceae,Prymnesiophyceae, Xanthophyceae, Raphidophyceae, Bacillariophyceae,Eustigmatophyceae, Crytophyceae, Euglenophyceae, Prasinophyceae, andChlorophyceae. Of particular interest are such phytosphere bacterialspecies as Pseudomonas syringae, Pseudomonas fluorescens, Serratiamarcescens, Acetobacter xylinum, Agrobacterium tumefaciens,Rhodopseudomonas spheroides, Xanthomonas campestris, Rhizobium melioti,Alcaligenes entrophus, and Azotobacter vinlandii; and phytosphere yeastspecies such as Rhodotorula rubra, R. glutinis, R. marina, R.aurantiaca, Cryptococcus albidus, C. diffluens, C. laurentii,Saccharomyces rosei, S. pretoriensis, S. cerevisiae, Sporobolomycesroseus, S. odorus, Kluyveromyces veronae, and Aureobasidium pollulans.Of particular interest are the pigmented microorganisms.

A wide variety of ways are available for introducing a B.t. geneexpressing a toxin into the microorganism host under conditions whichallow for stable maintenance and expression of the gene. One can providefor DNA constructs which include the transcriptional and translationalregulatory signals for expression of the toxin gene, the toxin geneunder their regulatory control and a DNA sequence homologous with asequence in the host organism, whereby integration will occur, and/or areplication system which is functional in the host, whereby integrationor stable maintenance will occur.

The transcriptional initiation signals will include a promoter and atranscriptional initiation start site. In some instances, it may bedesirable to provide for regulative expression of the toxin, whereexpression of the toxin will only occur after release into theenvironment. This can be achieved with operators or a region binding toan activator or enhancers, which are capable of induction upon a changein the physical or chemical environment of the microorganisms. Forexample, a temperature sensitive regulatory region may be employed,where the organisms may be grown up in the laboratory without expressionof a toxin, but upon release into the environment, expression wouldbegin. Other techniques may employ a specific nutrient medium in thelaboratory, which inhibits the expression of the toxin, where thenutrient medium in the environment would allow for expression of thetoxin. For translational initiation, a ribosomal binding site and aninitiation codon will be present.

Various manipulations may be employed for enhancing the expression ofthe messenger RNA, particularly by using an active promotor, as well asby employing sequences, which enhance the stability of the messengerRNA. The transcriptional and translational termination region willinvolve stop codon(s), a terminator region, and optionally, apolyadenylation signal. A hydrophobic "leader" sequence may be employedat the amino terminus of the translated polypeptide sequence in order topromote secretion of the protein across the inner membrane.

In the direction of transcription, namely in the 5' to 3' direction ofthe coding or sense sequence, the construct will involve thetranscriptional regulatory region, if any, and the promoter, where theregulatory region may be either 5' or 3' of the promoter, the ribosomalbinding site, the initiation codon, the structural gene having an openreading frame in phase with the initiation codon, the stop codon(s), thepolyadenylation signal sequence, if any, and the terminator region. Thissequence as a double strand may be used by itself for transformation ofa microorganism host, but will usually be included with a DNA sequenceinvolving a marker, where the second DNA sequence may be joined to thetoxin expression construct during introduction of the DNA into the host.

By a marker is intended a structural gene which provides for selectionof those hosts which have been modified or transformed. The marker willnormally provide for selective advantage, for example, providing forbiocide resistance, e.g., resistance to antibiotics or heavy metals;complementation, so as to provide prototropy to an auxotrophic host, orthe like. Preferably, complementation is employed, so that the modifiedhost may not only be selected, but may also be competitive in the field.One or more markers may be employed in the development of theconstructs, as well as for modifying the host. The organisms may befurther modified by providing for a competitive advantage against otherwild-type microorganisms in the field. For example, genes expressingmetal chelating agents, e.g., siderophores, may be introduced into thehost along with the structural gene expressing the toxin. In thismanner, the enhanced expression of a siderophore may provide for acompetitive advantage for the toxin-producing host, so that it mayeffectively compete with the wild-type microorganisms and stably occupya niche in the environment.

Where no functional replication system is present, the construct willalso include a sequence of at least 50 basepairs (bp), preferably atleast about 100 bp, and usually not more than about 5000 bp of asequence homologous with a sequence in the host. In this way, theprobability of legitimate recombination is enhanced, so that the genewill be integrated into the host and stably maintained by the host.Desirably, the toxin gene will be in close proximity to the geneproviding for complementation as well as the gene providing for thecompetitive advantage. Therefore, in the event that a toxin gene islost, the resulting organism will be likely to also lose thecomplementing gene and/or the gene providing for the competitiveadvantage, so that it will be unable to compete in the environment withthe gene retaining the intact construct.

A large number of transcriptional regulatory regions are available froma wide variety of microorganism hosts, such as bacteria, bacteriophage,cyanobacteria, algae, fungi, and the like. Various transcriptionalregulatory regions include the regions associated with the trp gene, lacgene, gal gene, the lambda left and right promoters, the tac promoter,the naturally-occurring promoters associated with the toxin gene, wherefunctional in the host. See for example, U.S. Pat. Nos. 4,332,898,4,342,832 and 4,356,270. The termination region may be the terminationregion normally associated with the transcriptional initiation region ora different transcriptional initiation region, so long as the tworegions are compatible and functional in the host.

Where stable episomal maintenance or integration is desired, a plasmidwill be employed which has a replication system which is functional inthe host. The replication system may be derived from the chromosome, anepisomal element normally present in the host or a different host, or areplication system from a virus which is stable in the host. A largenumber of plasmids are available, such as pBR322, pACYC184, RSF1010,pRO1614, and the like. See for example, Olson et al., (1982) J.Bacteriol. 150:6069, and Bagdasarian et al., (1981) Gene 16:237, andU.S. Pat. Nos. 4,356,270, 4,362,817, and 4,371,625.

The B.t. gene can be introduced between the transcriptional andtranslational initiation region and the transcriptional andtranslational termination region, so as to be under the regulatorycontrol of the initiation region. This construct will be included in aplasmid, which will include at least one replication system, but mayinclude more than one, where one replication system is employed forcloning during the development of the plasmid and the second replicationsystem is necessary for functioning in the ultimate host. In addition,one or more markers may be present, which have been describedpreviously. Where integration is desired, the plasmid will desirablyinclude a sequence homologous with the host genome.

The transformants can be isolated in accordance with conventional ways,usually employing a selection technique, which allows for selection ofthe desired organism as against unmodified organisms or transferringorganisms, when present. The transformants than can be tested forpesticidal activity.

Suitable host cells, where the pesticide-containing cells will betreated to prolong the activity of the toxin in the cell when the thentreated cell is applied to the environment of target pest(s), mayinclude either prokaryotes or eukaryotes, normally being limited tothose cells which do not produce substances toxic to higher organisms,such as mammals. However, organisms which produce substances toxic tohigher organisms could be used, where the toxin is unstable or the levelof application sufficiently low as to avoid any possibility of toxicityto a mammalian host. As hosts, of particular interest will be theprokaryotes and the lower eukaryotes, such as fungi. Illustrativeprokaryotes, both Gram-negative and -positive, includeEnterobacteriaceae, such as Escherichia, Erwinia, Shigella, Salmonella,and Proteus; Bacillaceae; Rhizobiceae, such as Rhizobium; Spirillaceae,such as photobacterium, Zymomonas, Serratia, Aeromonas, Vibrio,Desulfovibrio, Spirillum; Lactobacillaceae; Pseudomonadaceae, such asPseudomonas and Acetobacter; Azotobacteraceae, Actinomycetales, andNitrobacteraceae. Among eukaryotes are fungi, such as Phycomycetes andAscomycetes, which includes yeast, such as Saccharomyces andSchizosaccharomyces; and Basidiomycetes yeast, such as Rhodotorula,Aureobasidium, Sporobolomyces, and the like.

Characteristics of particular interest in selecting a host cell forpurposes of production include ease of introducing the B.t. gene intothe host, availability of expression systems, efficiency of expression,stability of the pesticide in the host, and the presence of auxiliarygenetic capabilities. Characteristics of interest for use as a pesticidemicrocapsule include protective qualities for the pesticide, such asthick cell walls, pigmentation, and intracellular packaging or formationof inclusion bodies; survival in aqueous enviroments; lack of mammaliantoxicity; attractiveness to pests for ingestion; ease of killing andfixing without damage to the toxin; and the like. Other considerationsinclude ease of formulation and handling, economics, storage stability,and the like.

Host organisms of particular interest include yeast, such as Rhodotorulasp., Aureobasidium sp., Saccharomyces sp., and Sporobolomyces sp.;phylloplane organisms such as Pseudomonas sp., Erwinia sp. andFlavobacterium sp.; or such other organisms as Escherichia,Lactobacillus sp., Bacillus sp., Streptomyces sp., and the like.Specific organisms include Pseudomonas aeruginosa, Pseudomonasfluorescens, Saccharomyces cerevisiae, Bacillus thuringiensis,Escherichia coli, Bacillus subtilis, Streptomyces lividans and the like.

The cell will usually be intact and be substantially in theproliferative form when treated, rather than in a spore form, althoughin some instances spores may be employed.

Treatment of the microbial cell, e.g., a microbe containing the B.t.toxin gene, can be by chemical or physical means, or by a combination ofchemical and/or physical means, so long as the technique does notdeleteriously affect the properties of the toxin, nor diminish thecellular capability in protecting the toxin. Examples of chemicalreagents are halogenating agents, particularly halogens of atomic no.17-80. More particularly, iodine can be used under mild conditions andfor sufficient time to achieve the desired results. Other suitabletechniques include treatment with aldehydes, such as formaldehyde andglutaraldehyde; anti-infectives, such as zephiran chloride andcetylpyridinium chloride; alcohols, such as isopropyl and ethanol;various histologic fixatives, such as Lugol iodine, Bouin's fixative,and Helly's fixative (See: Humason, Gretchen L., Animal TissueTechniques, W. H. Freeman and Company, 1967); or a combination ofphysical (heat) and chemical agents that preserve and prolong theactivity of the toxin produced in the cell when the cell is administeredto the host animal. Examples of physical means are short wavelengthradiation such as gamma-radiation and X-radiation, freezing, UVirradiation, lyophilization, and the like.

The cells generally will have enhanced structural stability which willenhance resistance to environmental conditions. Where the pesticide isin a proform, the method of inactivation should be selected so as not toinhibit processing of the proform to the mature form of the pesticide bythe target pest pathogen. For example, formaldehyde will crosslinkproteins and could inhibit processing of the proform of a polypeptidepesticide. The method of inactivation or killing retains at least asubstantial portion of the bio-availability or bioactivity of the toxin.

The cellular host containing the B.t. insecticidal gene may be grown inany convenient nutrient medium, where the DNA construct provides aselective advantage, providing for a selective medium so thatsubstantially all or all of the cells retain the B.t. gene. These cellsmay then be harvested in accordance with conventional ways.Alternatively, the cells can be treated prior to harvesting.

The B.t. cells may be formulated in a variety of ways. They may beemployed as wettable powders, granules or dusts, by mixing with variousinert materials, such as inorganic minerals (phyllosilicates,carbonates, sulfates, phosphates, and the like) or botanical materials(powdered corncobs, rice hulls, walnut shells, and the like). Theformulations may include spreader-sticker adjuvants, stabilizing agents,other pesticidal additives, or surfactants. Liquid formulations may beaqueous-based or non-aqueous and employed as foams, gels, suspensions,emulsifiable concentrates, or the like. The ingredients may includerheological agents, surfactants, emulsifiers, dispersants, or polymers.

The pesticidal concentration will vary widely depending upon the natureof the particular formulation, particularly whether it is a concentrateor to be used directly. The pesticide will be present in at least 1% byweight and may be 100% by weight. The dry formulations will have fromabout 1-95% by weight of the pesticide while the liquid formulationswill generally be from about 1-60% by weight of the solids in the liquidphase. The formulations will generally have from about 10² to about 10⁴cells/mg. These formulations will be administered at about 50 mg (liquidor dry) to 1 kg or more per hectare.

The formulations can be applied to the environment of the dipteranpest(s), e.g., plants, soil or water, by spraying, dusting, sprinkling,or the like.

Mutants of the novel isolate of the invention can be made by procedureswell known in the art. For example, an asporogenous mutant can beobtained through ethylmethane sulfonate (EMS) mutagenesis of the novelisolate. The mutants can be made using ultraviolet light andnitrosoguanidine by procedures well known in the art.

A smaller percentage of the asporogenous mutants will remain intact andnot lyse for extended fermentation periods; these strains are designatedlysis minus (-). Lysis minus strains can be identified by screeningasporogenous mutants in shake flask media and selecting those mutantsthat are still intact and contain toxin crystals at the end of thefermentation. Lysis minus strains are suitable for a cell fixationprocess that will yield a protected, encapsulated toxin protein.

To prepare a phage resistant variant of said asporogenous mutant, analiquot of the phage lysate is spread onto nutrient agar and allowed todry. An aliquot of the phage sensitive bacterial strain is then plateddirectly over the dried lysate and allowed to dry. The plates areincubated at 30° C. The plates are incubated for 2 days and, at thattime, numerous colonies could be seen growing on the agar. Some of thesecolonies are picked and subcultured onto nutrient agar plates. Theseapparent resistant cultures are tested for resistance by cross streakingwith the phage lysate. A line of the phage lysate is streaked on theplate and allowed to dry. The presumptive resistant cultures are thenstreaked across the phage line. Resistant bacterial cultures show nolysis anywhere in the streak across the phage line after overnightincubation at 30° C. The resistance to phage is then reconfirmed byplating a lawn of the resistant culture onto a nutrient agar plate. Thesensitive strain is also plated in the same manner to serve as thepositive control. After drying, a drop of the phage lysate is plated inthe center of the plate and allowed to dry. Resistant cultures showed nolysis in the area where the phage lysate has been placed afterincubation at 30° C. for 24 hours.

Following are examples which illustrate procedures, including the bestmode, for practicing the invention. These examples should not beconstrued as limiting. All percentages are by weight and all solventmixture proportions are by volume unless otherwise noted.

EXAMPLE 1 Culturing of the Novel B.t. Isolate

A subculture of the novel B.t. isolate, or mutants thereof, can be usedto inoculate the following medium, a peptone, glucose, salts medium.

    ______________________________________                                        Bacto Peptone          7.5    g/l                                             Glucose                1.0    g/l                                             KH.sub.2 PO.sub.4      3.4    g/l                                             K.sub.2 HPO.sub.4      4.35   g/l                                             Salt Solution          5.0    ml/l                                            CaCl.sub.2 Solution    5.0    ml/l                                            Salts Solution (100 ml)                                                       MgSO.sub.4.7H.sub.2 O  2.46   g                                               MnSO.sub.4.H.sub.2 O   0.04   g                                               ZnSO.sub.4.7H.sub.2 O  0.28   g                                               FeSO.sub.4.7H.sub.2 O  0.40   g                                               CaCl.sub.2 Solution (100 ml)                                                  CaCl.sub.2.2H.sub.2 O  3.66   g                                               pH 7.2                                                                        ______________________________________                                    

The salts solution and CaCl₂ solution are filter-sterilized and added tothe autoclaved and cooked broth at the time of inoculation. Flasks areincubated at 30° C. on a rotary shaker at 200 rpm for 64 hr.

The above procedure can be readily scaled up to large fermentors byprocedures well known in the art.

The B.t. spores and/or crystals, obtained in the above fermentation, canbe isolated by procedures well known in the art. A frequently-usedprocedure is to subject the harvested fermentation broth to separationtechniques, e.g., centrifugation.

EXAMPLE 2 Activity of B.t. PS192N1 Against Aedes aegypti

Aedes aegypti, the yellow fever mosquito, is used as an indicator ofmosquito activity. The bioassay is performed on a spore and crystalsuspension or a suspension of purified crystals. Dilutions of thesuspension are added to water in a small cup. Fourth instar larvae areadded, and mortality is read after 48 hours.

B.t. PS192N1 has an LC₅₀ of 10 μg protein/ml suspension against Aedesaegypti.

EXAMPLE 3 Insertion of Toxin Genes Into Plants

The novel genes, obtainable from the novel B.t. isolate of theinvention, coding for the novel insecticidal toxin, as disclosed herein,can be inserted into plant cells using the Ti plasmid from Agrobactertumefaciens. Plant cells can then be caused to regenerate into plants(Zambryski, P., Joos, H., Gentello, C., Leemans, J., Van Montague, M.and Schell, J [1983] Cell 32:1033-1043). A particularly useful vector inthis regard is pEND4K (Klee, H. J., Yanofsky, M. F. and Nester, E. W.[1985] Bio/Technology 3:637-642). This plasmid can replicate both inplant cells and in bacteria and has multiple cloning sites for passengergenes. The toxin gene, for example, can be inserted into the BamHI siteof pEND4K, propagated in E. coli, and transformed into appropriate plantcells.

EXAMPLE 4 Cloning of Novel B. thuringiensis Genes Into Baculoviruses

The novel genes, obtainable from the novel B.t. isolate of theinvention, can be cloned into baculoviruses such as Autographacalifornica nuclear polyhedrosis virus (AcNPV). Plasmids can beconstructed that contain the AcNPV genome cloned into a commercialcloning vector such as pUC8. The AcNPV genome is modified so that thecoding region of the polyhedrin gene is removed and a unique cloningsite for a passenger gene is placed directly behind the polyhedrinpromoter. Examples of such vectors are pGP-B6874, described by Pennocket al. (Pennock, G. D., Shoemaker, C. and Miller, L. K. [1984] Mol.Cell. Biol. 4:399-406), and pAC380, described by Smith et al. (Smith, G.E., Summers, M. D. and Fraser, M. J. [1983] Mol Cell. Biol.3:2156-2165). The gene coding for the novel protein toxin can bemodified with BamHI linkers at appropriate regions both upstream anddownstream from the coding region and inserted into the passenger siteof one of the AcNPV vectors.

I claim:
 1. A biologically pure culture of Bacillus thuringiensisPS192N1, having the identifying dipteran insect pest controllingcharacteristic of culture deposit NRRL B-18721, or mutants thereof whichhave the dipteran insect pest controlling characteristics of saiddeposit.